Aerobic organisms possess antioxidant defense systems that modulate reactive oxygen species (ROS). The superoxide dismutase (SODs) catalyze the dismutation of superoxide radicals into hydrogen peroxide and molecular oxygen. Hydrogen peroxide is further detoxified by catalase and glutathione peroxidase (Halliwell and Gutterbridge, 1989). Three distinct SODs are found in human cells; a monodimeric cytosolic CuZnSOD (McCord and Fridovich, 1969); an extracellular homotetrameric glycosylated CuZnSOD (ECSOD) (Marklund, 1982); and a mitochondrial matrix homotetrameric MnSOD (Weisiger and Fridovich, 1973).
Accumulating data suggest that MnSOD constitutes one of the major cellular defense mechanism against the toxic effects of agents that cause oxidative stress. It has been demonstrated that MnSOD knockout mice develop cardiomyopathy and neonatal lethality, whereas independent disruption of the genes for CuZnSOD and ECSOD isoenzymes result in viable, normal mice under nonstress conditions (reviewed in Yen and St. Clair, 1997). Furthermore, transgenic mice expressing human MnSOD in the mitochondria are protected from oxygen-induced cardiac injury (Yen et al., 1996), and ischemia-induced brain injury (Keller et al., 1998).
Numerous studies using gene transfection have demonstrated that transfection of MnSOD into tumor cells reverse the malignant phenotypes of tumor cells, suggesting that MnSOD functions to suppress tumorigenicity (reviewed in St. Clair et al., 1997). Transfection of human MnSOD cDNA into mouse fibroblasts prevents radiation-induced neoplastic transformation (St. Clair et al. 1992). Expression of the human MnSOD gene in mouse C3H10T1/2 cells enhances cellular differentiation upon treatment with 5-azacytidine (St. Clair et al. 1994). The malignant phenotype of human melanoma cells was suppressed by introduction of human chromosome 6 where the MnSOD gene is located (Trent et al. 1990) or transfection of a human MnSOD cDNA (Church et al., 1993). Overexpression of MnSOD suppressed the malignant phenotypes of human breast cancer cells (Li et al. 1995), human glioma cells (Zhong et al., 1997), and mouse epidermal cells (Amstad et al., 1997). The number of cells required to produce tumors in syngenic mice was markedly increased for the MnSOD-transfected murine fibrosarcoma cells lines (MnSOD-Fsa-II) compared to the vector-transfected control cells (St. Clair et al., 1997). The frequency of metastases was reduced in syngenic mice carrying the MnSOD transfected-FsaII cells compared to the mice bearing the control FsaII cells (Stafford et al., 1994). Furthermore the radiation dose required to control one-half of the irradiate tumor (TCD50) was greatly reduced when the MnSOD-FsaII cells were transplanted and irradiated in vivo under hypoxic conditions (Urano et al., 1995). Taken together, the evidence from these studies supports a hypothesis proposed by Oberley and Oberley (1984) that MnSOD plays an important role in the prevention of cancer development.
It has been shown that many types of human cancer cells have reduced MnSOD activity compared to their appropriate normal counterpart cells; (oberley and Buettner, 1979). The reduced level of MnSOD activity in human cancer cells is not due to a defect in the primary structure of the MnSOD protein, a change in the dosage of the MnSOD gene, or a decrease in the stability of MnSOD mRNA in tumor cells, but rather is due to defects in the expression of the gene (St. Clair and Holland, 1991).
The present inventors previously cloned and sequenced the entire human MnSOD gene, including a 0.7 kb 5' flanking region, from a genomic library obtained from normal human lung fibroblast cells. The gene is characterized by the lack of TATA or CAAT box regulatory elements and the presence of a GC-rich region containing multiple SP-1 binding sites (Wan et al., 1994).
Prior research has failed to elucidate the cause for reduced expression of MnSOD in tumor cells. The present inventors have undertaken extensive research in order to solve this problem, and have accomplished this result by discovering several highly conserved mutations in the promoter region of the MnSOD gene.
In order to elucidate the cause for the reduced expression of human MnSOD in tumor cells, the present inventors have now further sequenced the 5' flanking region of the human SOD gene and compared that to the 5' flanking region of the human MnSOD gene from several tumor cell lines. The results demonstrate three heterozygous mutations n the promoter region of the human MnSOD gene in 5 of 14 tumor cell lines examined. Significantly these mutations were conserved amongst 3 of 5 colon cancer cell lines studied. The effect of these mutations on the transcription activity of the human MnSOD promoter was also determined by means of a reporter gene constructs. These results demonstrated markedly reduced gene expression when compared to transcriptional activation of the normal, wild-type MnSOD promoter.
An object of the present invention is to provide a diagnostic kit and a diagnostic method for assaying the presence of superoxide dismutase gene mutations associated with decreased enzyme activity seen in many diseases. Specifically, this invention aims at providing a diagnostic kit and method for certain cancers associated with reduced MnSOD expression. Another object of the present invention is to provide a diagnostic test and a diagnostic kit for Amylotrophic Lateral Sclerosis, ALS, which his also associated with reduced MnSOD activity.
Another objection of the present invention is to provide a therapeutic method targeted at disease associated with decreased MnSOD activity, and more specifically targeted at cancer and ALS.